Methicillin-resistant Staphylococcus aureus (MRSA) is the causative agent of a wide diversity of diseases ranging from benign skin infection to life-threatening diseases such as endocarditis, osteomyelitis, rhinosinusitis, sepsis, necrotizing pneumonia. Resistant to beta-lactam antibiotics and now most often multi-resistant, MRSA is disseminated within healthcare institutions and the community making effective antibiotic therapy problematic. Colonization with this pathogen is a risk factor for eventual MRSA clinical infection, which is associated with higher healthcare costs and poor outcomes. Over the last few decades it has become a major pathogen worldwide. To get MRSA dissemination under control and rapidly diagnose infection, an improved simple, fast, and reliable diagnostic tool remains urgently needed. Current diagnostics are either excessively time consuming or suffer from suboptimal sensitivity/specificity. The objective of this application is to develop and validate methodology for a fast, highly accurate identification of MRSA that will utilize novel molecular diagnostic methods. The rationale behind the proposed research is to utilize genomic DNA from specimens (nasal swabs as the initial selection) with the subsequent enrichment of the target (S. aureus) during the unique S. aureus sequence(s) hybridization to probe(s) immobilized on magnetic beads with subsequent formation of novel DNA structures and then detection of the specific S. aureus resistance gene (mecA). To accomplish the objectives of this application, we will pursue three specific aims: 1) Develop and optimize the molecular approach (reagents, protocol and procedures) to result in specificity of captured microorganism DNA that will be unique target genes for S. aureus in direct specimens;2) refine our rapid hybridization approach for separation of S. aureus DNA from other species in an analyte with subsequent detection for methicillin resistance;3) evaluate our method by comparison to culture and real-time (q)-PCR assays. At the completion of our research, we expect to have developed methodology that will specifically and quickly identify MRSA from nasal swab utilizing affordable approaches. To further this effort, we will team with investigators at Nanosphere Inc. to integrate our algorithm with their nanotechnology and detection platform and evaluate the feasibility for developing a reliable, cost-effective diagnostic system for MRSA suitable for a variety of healthcare settings. Our long term goal is to develop an easy to use, highly accurate diagnostic tool for earlier detection of pathogen specific microbial DNA in clinical samples. PUBLIC HEALTH RELEVANCE: Resistant to beta-lactam antibiotics and now most often multi-resistant to many antimicrobial agent classes, methicillin-resistant Staphylococcus aureus (MRSA) is a worldwide pathogen that has disseminated within healthcare institutions and the community - making effective antibiotic therapy problematic. The availability of a rapid and very reliable MRSA screening / diagnostic method and determining its value in daily practice is of great importance since accuracy of current diagnostic assays is still limited by the fact of their target design. Our innovative methodology will improve patient care by developing a novel diagnostic methodology that enhances specificity over current tests for both infection control surveillance and real-time pathogen detection during infection, thus lowering the potential risk of severe disease and death as well as improve the appropriate (over and under) use of antimicrobial agents.